The present invention relates to stable and preserved erythropoietin pharmaceutical compositions that provide convenience of use and ease of administration.
Erythropoietin is a glycoprotein hormone that stimulates the formation of red blood cells. Erythropoietin is produced in the kidney, and once produced, it circulates to the bone marrow where it stimulates the conversion of primitive precursor cells into proerythroblasts which subsequently mature into red blood cells. In the normal healthy state, erythropoietin is present in very low concentrations in plasma, i.e., about 0.01 to 0.03 U/ml, but when the level of oxygen in transport is reduced, a condition known as hypoxia, the kidney produces more erythropoietin. Hypoxia can be the result of loss of large amounts of blood, destruction of red blood cells by radiation, or exposure to high altitudes. In addition, various forms of anemia cause hypoxia since red blood cells are responsible for oxygen transport in the body. In the normal state, an increased level of erythropoietin stimulates the production of new red blood cells thereby raising the level of oxygen and reducing or eliminating the hypoxic condition.
In contrast to this correction of hypoxia which occurs normally, patients with chronic renal failure ("CRF") have limited or no production of erythropoietin, and consequently, do not produce sufficient red blood cells. As the normal life span for red blood cells is about 120 days, such patients become increasing anemic with time. Prior to the development of recombinant erythropoietin, patients with chronic renal failure often had to undergo regular blood transfusions to maintain a minimum level of red blood cells.
The production of human erythropoietin outside the body using recombinant DNA technology is described in U.S. Pat. No. 4,703,008 to Lin and assigned to Amgen Inc., the assignee hereof. Recombinant human erythropoietin (rEPO) is produced by mammalian cells into which the human erythropoietin gene has been introduced. These cells produce a polypeptide product which has the identical amino acid sequence of isolated natural erythropoietin. Moreover, this rEPO has been shown to have the same biological effects as endogenous erythropoietin even though rEPO is known to have a different carbohydrate composition than naturally occurring erythropoietin (Strickland et al, J. Cell. Biochem., Supplement 16D, P324, 1992). rEPO has proven to be extremely effective in treating various forms of anemias, especially that associated with end stage renal disease. The administration of rEPO, as replacement therapy for the missing protein, significantly reduces or eliminates the need for blood transfusions in most CRF patients. Similarly, rEPO has proven to be effective in treating other types of anemia including that associated with the treatment of AIDS using AZT.
Amgen has sold rEPO in the United States for research purposes since 1985, and for therapeutic purposes since June 1989. Amgen manufactures rEPO which has been given the proper name of Epoetin alfa. Epoetin alfa is sold by Amgen under the trademark EPOGEN.RTM. and by Ortho Pharmaceutical under the trademark PROCRIT.RTM.. Epoetin alfa is formulated and sold as an aqueous solution of rEPO containing sodium citrate buffer to maintain the solution at about pH 7, human serum albumin (HSA), sodium chloride, and water for injection, USP. The use of HSA in rEPO formulations was first disclosed in Lin, U.S. Pat. No. 4,703,008, Col. 35 lines 17-20. The current formulation of Epoetin alfa contains no antimicrobial preservative and is sold in vials designated as "single-use" and containing 2,000, 3,000, 4,000 or 10,000 U/ml of rEPO. Epoetin alfa is an injectable product approved for administration by either intravenous or subcutaneous routes.
In evaluating the use of Epoetin alfa, certain needs have been discovered regarding its formulation and delivery. First, it has been reported that some patients experience local discomfort or stinging upon the subcutaneous administration of Epoetin alfa. In a study reported by Frenken, L., et al. in British Medical Journal, 1991; 303:288, on the use of Exprex.RTM., a rEPO product produced by Cilag GMBH which is formulated identically to Epoetin alfa, a number of patients experienced a burning or itching after subcutaneous injection. Ameliorating this stinging effect without decreasing the ease of administration is highly desired. Second, being an injectable product, some users and caregivers would greatly benefit by being able to draw several doses from a single vial, i.e., a multi-dose product as opposed to a single-dose. In order for a pharmaceutical product to be multi-dose it must contain an antimicrobial agent in order to kill or inhibit the growth of any microbes which may inadvertently be introduced into the container. This prevents the growth and subsequent administration of such microbes to the patient. For some, a multi-dose rEPO would reduce handling and inventory while eliminating waste of a valuable product. The combination of a non-sting and multi-dose, ready-to-use solution formulation is highly desirable and meets several needs of the patient and caregiver.
In order to be commercially useful, the pharmaceutical composition must be stable. Others have suggested different formulations of rEPO which are reported to be stable, but none contain a preservative thereby providing multi-dose capability. The literature suggests and illustrates the relative instability of erythropoietin in solution and the sensitivity of erythropoietin to various additives.
U.S. Pat. No. 4,806,524 to Kawaguchi and Shimoda discloses lyophilized and aqueous solution preparations containing erythropoietin and one or more stabilizers. Kawaguchi et al. report the need for stabilizers since "erythropoietin is an instable substance and the compound purified to a clinically acceptable level and formulated in a trace amount (.apprxeq. a few .mu.g) that is suitable for a single dosage will be easily inactivated by such environmental factors as temperature and humidity." (Col.1, lines 15-20) Kawaguchi et al. reported testing erythropoietin derived from human urine with a long list of various "stabilizers". The results of stability for lyophilized formulation show fair stability, but the results for aqueous solutions show that the solutions are very instable. After only one week at 25.degree. C., the solution formulations showed losses in residual activity of about 30% or more as measured by in vivo testing. (See Table in U.S. Pat. No. 4,806,524). The lyophilized forms showed greater stability after being exposed to 37.degree. C. for one month, while still in the lyophilized form, but many of the formulations showed losses of 5-10% or more. The results of Kawaguchi et al. confirm their statement that erythropoietin is an "instable substance" especially in solution form and further reveal that when combined with known stabilizers, the resulting stability of the erythropoietin is varied and unpredictable.
In U.S. Pat. No. 4,879,272, Shimoda and Kawaguchi show an increase in recovery of erythropoietin by using additives to reduce the adsorption of erythropoietin onto the wall of a container. The results shown in the Table, using urinary erythropoietin with different additives and measured after only two hours at room temperature, however, report the rapid and varied loss of erythropoietin in solution. The values reported range from 69.2-98.8% recovery of erythropoietin for the solutions containing additives. While the results compare favorable to the recovery of only 16.5% with no additive, generally the loss is still very significant. However, as described in Experiments 1 and 2, this data reflects only the level of a radioactive labeled product and reveal nothing about the loss of erythropoietin to decomposition or whether the remaining product is active. As in U.S. Pat. No. 4,806,524, the data again reflects the different effects of different additives on erythropoietin.
Further, U.S. Pat. No. 4,992,419 to Woog et al. disclose lyophilized erythropoietin preparations that include a buffer, urea, a detergent and one or more of various amino acids. In addition, various other additives are suggested. In fact, Woog et al. state that the urea and amino acids are "decisive for stabilization." (Col. 2, lines 43-44). Urea is used in rather high amounts relative to the amount of erythropoietin, i.e., 5 to 50 g./liter and preferably 10-15 g./liter. The specified amino acids used in the formulation are glycine, L-alanine, L-arginine, L-isoleucine, L-leucine, L-phenylalanine, L-glutamic acid and L-threonine. In addition, a detergent or wetting agent are added to the composition to reduce the adhesion of erythropoietin to the walls of the vessel. At Col. 4, lines 3-8, Woog et al. report that aqueous solutions of erythropoietin having these additives are stable for about 1 year at 0.degree. C. and only a few months at ambient temperatures. Although Woog et al. disclose various additives to be used in erythropoietin solutions, no suggestion is given concerning the use of a preservative.
In contrast to the formulations described in the above-cited U.S. Patents, Epoetin alfa has proven to be quite stable in solution form and it has an approved shelf-life of 24 months under refrigerated conditions, 2.degree.-8.degree. C., as approved by the United States Food and Drug Administration. Moreover, as a liquid, ready-to-use formulation, Epoetin alfa is more convenient to administer than a lyophilized formulation which must be reconstituted prior to administration. This reconstitution procedure is time consuming for the medical personnel involved, and it poses the risk of mishandling and improper reconstitution. For these reasons, among others, solution formulations are generally preferred over lyophilized ones.
Aside from the discussion above, very little has been reported concerning the stability of erythropoietin in solution, and nothing is disclosed concerning preserved formulations of erythropoietin. Also, since erythropoietin is one of the early recombinant proteins to be produced, nothing specific can be derived from the use of preservatives with other proteins that would suggest any particular preserved formulation for erythropoietin. See, e.g., Geigert, J., "Overview of the Stability and Handling of Recombinant Protein Drugs," Journal of Parenteral Science & Technology, Vol. 43, No. 5, 220-224 (1989). In that article, Geigert discusses the recombinant protein drugs approved for marketing by the FDA as of March 1989. Of the marketed recombinant drugs, two were sold as liquids and four as lyophilizates. (See Table VI of Geigert). The two liquid products have approved expiration dates of one and two years at 2.degree.-8.degree. C. Geigert also reports that for the lyophilized products reconstituted with a bacteriostat in the solution, the expiration dating for those solutions ranged from 7-30 days at 2.degree.-8.degree. C. (See Table VII). Moreover, Geigert reports that IL-2, a product which at this time was undergoing clinical trials, is not compatible with bacteriostatic agents.
Nothing can be drawn from the literature concerning a stable and preserved erythropoietin formulation. A need still exists for a such a formulation that would provide the benefits of a multi-dose solution formulation, and additionally that would avoid the discomfort or stinging sometimes associated with subcutaneous injection of certain solution formulations. The present invention provides such a formulation. In contrast to the formulations suggested by the above cited patents, the formulations of the present invention are multi-dose and stable for extended periods of time.